N-phosphonomethylglycine, also known as glyphosate, is a well-known herbicide that has activity on a broad spectrum of plant species. Glyphosate is the active ingredient of Roundup® (Monsanto Co., St Louis, Mo.), a herbicide having a long history of safe use and a desirably short half-life in the environment. When applied to a plant surface, glyphosate moves systemically through the plant. Glyphosate is phytotoxic due to its inhibition of the shikimic acid pathway, which provides a precursor for the synthesis of aromatic amino acids. Glyphosate inhibits the class I 5-enolpyruvyishikimate-3-phosphate synthase (EPSPS) found in plants and some bacteria. Glyphosate tolerance in plants can be achieved by the expression of a modified class I EPSPS that has lower affinity for glyphosate, yet still retains its catalytic activity in the presence of glyphosate (U.S. Pat. Nos. 4,535,060, and 6,040,497). “Tolerant” or “tolerance” refers to a reduced effect of an agent on the growth and development, and yield of a plant, in particular, tolerance to the phytotoxic effects of a herbicide, especially glyphosate.
Enzymes, such as, class II EPSPSs have been isolated from bacteria that are naturally resistant to glyphosate and when the enzyme is expressed as a transgene in plants provides glyphosate tolerance to the plants (U.S. Pat. Nos. 5,633,435 and 5,094,945). Enzymes that degrade glyphosate in plant tissues (U.S. Pat. No. 5,463,175) are also capable of conferring plant tolerance to glyphosate. DNA constructs that contain the necessary genetic elements to express the glyphosate resistant enzymes or degradative enzymes create chimeric transgenes useful in plants. Such transgenes are used for the production of transgenic crops that are tolerant to glyphosate, thereby allowing glyphosate to be used for effective weed control with minimal concern of crop damage. For example, glyphosate tolerance has been genetically engineered into corn (U.S. Pat. No. 5,554,798), wheat (Zhou et al. Plant Cell Rep. 15:159-163, 1995), soybean (WO 9200377) and canola (WO 9204449). The transgenes for glyphosate tolerance and transgenes for tolerance to other herbicides, for example the bar gene (Sh.Bar) may be included in DNA constructs for use as a selectable marker for plant transformation (present invention pMON81519; and Toki et al. Plant Physiol., 100:1503-1507, 1992; Thompson et al. EMBO J. 6:2519-2523, 1987; phosphinothricin acetyltransferase DeBlock et al. EMBO J., 6:2513-2522, 1987, glufosinate herbicide) are also useful as selectable markers or scorable markers and can provide a useful phenotype for selection of transgenic plants when the marker gene is linked with other agronomically useful traits.
Development of herbicide-tolerant crops has been a major breakthrough in agriculture biotechnology as it has provided farmers with new weed control methods. One enzyme that has been successfully engineered for resistance to its inhibitor herbicide is class I EPSPS. Variants of class I EPSPS have been isolated (Pro-Ser, U.S. Pat. No. 4,769,061; Gly-Ala, U.S. Pat. No. 4,971,908; Gly-Ala, Gly-Asp, U.S. Pat. No. 5,310,667; Gly-Ala, Ala-Thr, U.S. Pat. No. 5,8866,775) that are resistant to glyphosate. However, many EPSPS variants either do not demonstrate a sufficiently high Ki for glyphosate or have a Km for phosphoenol pyruvate (PEP) too high to be effective as a glyphosate resistance enzyme for use in plants (Padgette et. al, In “Herbicide-resistant Crops”, Chapter 4 pp 53-83. ed. Stephen Duke, Lewis Pub, CRC Press Boca Raton, Fla. 1996). However, one class I EPSPS variant, T102I/P106S (TIPS) that is operably linked to a heterologous promoter has been shown to provide glyphosate tolerance to transgenic maize plants (U.S. Pat. No. 6,040,497). A glyphosate tolerant EPSPS has also been isolated from the weed Eleusine indica [WO 01/66704].
There is a need in the field of plant molecular biology for a diversity of genes that can provide a positive selectable marker phenotype. In particular, glyphosate tolerance is used extensively as a positive selectable marker in plants and is a valuable phenotype for use in crop production. The stacking and combining of existing transgene traits with newly developed traits is enhanced when distinct positive selectable marker genes are used. The marker genes provide either a distinct phenotype, such as, antibiotic or herbicide tolerance, or a molecular distinction discernable by methods used for DNA detection. The transgenic plants can be screened for the stacked traits by analysis for multiple antibiotic or herbicide tolerance or for the presence of novel DNA molecules by DNA detection methods. The present invention provides DNA and protein compositions of glyphosate resistant variant class I EPSP synthases. The present invention also provides DNA constructs useful in plants and transgenic plants that exhibit glyphosate tolerance.